1. Field of the Invention
This invention relates to the cryopreservation of both harvested tissue and cultured tissue equivalents made using in vitro technology. By use of the cryopreservation technology, either cryopreserved harvested tissue or cryopreserved cultured tissue may be stored for indefinite periods of time prior to use. The cultured tissue is an in vitro model of the equivalent human tissue, which, when retrieved from storage, can be used for transplantation or implantation, in vivo, or for screening compounds in vitro.
2. Brief Description of the Background of the Invention
In vitro technology has developed tissue equivalents for the purposes of in vitro testing or in vivo grafting for wound repair. Methods of producing such tissue equivalents are disclosed in U.S. Pat. Nos. 4,485,096, 4,604,346, 4,835,102 and 5,374,515 and are incorporated herein by reference.
The shelf life of living tissues is limited and, subsequently, their window of use is short, resulting in much waste. There is a need to preserve such tissues for extended periods of time, as in shipping and storage, until their use. The development of a cryopreservation method would extend the window of use indefinitely, ease shipping and allow for the maintenance of an inventory. To enable an inventory of tissue at burn care centers and hospitals is also desirable. Other advantages are that samples can be retained from different stages of the manufacturing cycle for quality control archives and larger production batches can be made as they can be maintained in a frozen state.
Currently, the storage time of biological materials is extended by cooling to "cryogenic" temperatures. The transition from the liquid into the solid state by lowering the temperature of the system can take place either as crystallization (ice), involving an orderly arrangement of water molecules, or as vitrification or amorphization (glass formation), in the absence of such an orderly arrangement of crystalline phase. The challenge for a cryobiologist is to bring cells to cryogenic temperatures and then return them to physiological conditions without injuring them.
There are two basic approaches to cryopreservation of cells and tissues: freeze-thaw and vitrification. In freeze-thaw techniques, the extracellular solution is frozen (i.e., in crystalline form), but steps are taken to minimize the intracellular ice formation. In vitrification procedures, there is an attempt to prevent ice formation throughout the entire sample. The former approach is problematic in that if ice crystals are formed inside the cells, they are detrimental to cell viability upon thawing. However, cells could survive a freeze-thaw cycle if they are cooled at controlled rates in the presence of non-toxic levels of cryoprotectants. The latter approach of vitrification seeks to avoid potentially damaging affects of intra- and extracellular ice by depressing ice formation using very high concentrations of solutes and/or polymers. However, the cell damage may occur to long exposure to toxic levels of these additives required for vitrification.
Cryoprotectants protect living cells from the stresses involved in the freezing process. One way cryoprotectants protect cells is by diluting the salt that becomes increasingly concentrated in the unfrozen solution as water is transformed to ice. The amount of ice is dictated by the temperature and initial composition of the solution; whereas the amount of unfrozen fraction is a function of temperature only. Cryoprotectants have several other functions. An important one is that they usually reduce the intracellular ice formation temperatures. Another function is that they stabilize membranes and proteins.
All solutions will supercool below their freezing point until they find a random nucleation site for crystal formation. When cryopreserving by a freeze-thaw method, ice formation in the extracellular medium should be deliberately initiated by seeding at low degrees of supercooling. If ice formation is not induced by seeding, ice will form spontaneously when the solution is cooled sufficiently far below its equilibrium freezing point. Because this process is random in nature, ice formation will occur at random, unpredictable temperatures; consequently, survival rates will be highly variable between repeated trials with the same freezing protocol. Furthermore, the extremely rapid crystallization which results when ice forms in a highly supercooled solution can cause damage to cells and tissues. Moreover, it has been shown that if extracellular ice formation is initiated at high degrees of supercooling, the probability of intracellular ice formation is drastically increased. This phenomenon results from the delayed onset of freeze-induced cell dehydration, which results in increased retention of intracellular water, and thus higher likelihood of ice formation in the cell.
Once the extracellular ice is seeded and the sample is surrounded by the ice phase, it is necessary to cool the sample to a cryopreserved state. The cooling step is one of the most critical steps in a freeze-thaw protocol. Due to the formation of ice, i.e., pure water, the partially frozen extracellular solution is more concentrated than the intracellular compartment. As a consequence, the cell will dehydrate by losing water in an attempt to restore thermodynamic equilibrium. As the system cools, more extracellular ice is generated and the concentration of solutes rises and forces the cells to dehydrate further. There are three characteristics of the cells that control their rate of dehydration. One is the cell membrane water permeability; the lower the water permeability, the longer it takes for the cells to dehydrate. Another is the temperature dependence of the cell membrane water permeability; all cells decrease their water permeability with decreasing temperatures. The final is cell size; larger cells take longer to dehydrate than smaller cells. Given that each cell type may have drastically different characteristics, the optimal cryopreservation conditions can vary by orders of magnitude for different cell types.
Although the exact mechanisms of cell damage during cryopreservation has not yet been completely elucidated, characteristic survival signatures generated by measuring cell survival as a function of cooling rate appear to be qualitatively similar for all cell types and displays an inverted U shaped curve. Cell survival is low at very slow and very fast cooling rates, and there is an intermediate cooling rate yielding optimal survival. Even though the optimal cooling rate and the width of the curve can vary drastically for different cell types, the qualitative behavior appears to be universal. Faster cooling rates do not allow cells enough time to dehydrate and cells form ice internally. Cell injury at fast cooling rates is attributed to intracellular ice formation. At slow rates of cooling, cell injury is thought to be due to the effects of exposure to highly concentrated intra- and extracellular salt and cryoprotectant solutions or to the mechanical interactions between cells and the extracellular ice.
It is necessary to dehydrate the cells as much as possible before they cross the intracellular ice nucleation curve. It is at this point that practically all water remaining in the cell will nucleate and form ice. It is impractical to determine the exact temperature where this will happen but it is approximately -40.degree. C. to -50.degree. C. when the cells are slowly frozen in the presence of 1M to 2M concentrations of cryoprotectants. It is important to note that the amount of water that turns to ice inside a cell at this point may be innocuous when frozen, but if not thawed fast enough, it will expand and kill the cell upon thawing. (The Biophysics of Organ Cryopreservation, Pg. 117-140, edited by David E. Pegg and Armand M. Karow, Jr. NATO ASI Series A: Life Sciences Vol. 147 1987 Plenum Press, New York 233 Spring St., New York, N.Y. 10013).
Before the development of a commercially viable skin equivalent, cadaver skin was used for the purposes of grafting. Cryopreservation protocols were developed so that burn centers and hospitals could maintain skin banks. A number of different protocols were developed utilizing different cryoprotectants, freeze rates, packaging formats and storage conditions. Most researchers agreed upon a fast thaw protocol. The success or failure of the protocol was measured either by graft take to a wound bed or by cell viability assay.
In U.S. Pat. No. 3,842,831 to Beisang is disclosed a method for the cryopreservation of cadaver skin patches. The method involves the attachment of the cadaver skin to a loosely woven scrim or backing and, together, the skin patches and the scrim are rolled prior to freezing. No cryoprotectant is employed, though the inventors suggest the use of either glycerin or DMSO. The freezing protocol employs a fast uncontrolled (fixed temperature) freeze rate protocol to a cryogenic temperature of -70.degree. C.
May S R and F A DeClement, Skin Banking Methodology, 17, 33-45 (1980), performed an evaluation of packaging geometry and cooling and warming rates using dermatome cadaver skin. The results suggested that cadaver skin be flat, rather than rolled, and that a slower controlled rate of freezing be employed.
U.S. Pat. No. 5,145,770 to Tubo discloses a cryopreservation method for keratinocyte sheets that employs a cryoprotectant of a non-cell penetrating agent, such as dextran, and a cell penetrating reagent, such as glycerol, with a cooling rate of about -1.degree. C./minute. Similarly, EP 0 364 306 to Chao et al, discloses a method for cryopreserving a sheet of living, cultured epithelial cells but utilizing both DMSO and glycerol as a cryoprotectant and a freezing protocol of preferably -1.degree. C./minute.
U.S. Pat. No. 5,298,417 to Cancedda et al, discloses a cryopreservation protocol developed for single layer constructs such as epithelial sheets prepared as described in U.S. Pat. Nos. 4,016,036, 4,304,866 and 4,456,687. Epidermal sheets were incubated with a cryoprotectant of either 8-15% glycerol or DMSO and were cryopreserved by employing a controlled rate protocol where the cooling rate is slower at the start than at the end of the protocol and is characterized by an increase in temperature before the culmination of the freezing procedure.
A method for the cryoprotection of dermal fibroblasts in a collagen gel was investigated by Teasdale et al, Burns, 19 (5) 406-410 (1993). Teasdale determined that optimum cell viability could be obtained by freezing at -0.5.degree. C./minute with DMSO as a cryoprotectant.
Nanchahal et al., "Cultured composite skin grafts: Biological skin equivalents permitting massive expansion," The Lancet, 2 (8565), 191-193 (Jul. 22, 1989), discusses a technique for storage of composite cultured tissue grafts utilizing a cryoprotectant of 15% glycerol and 10% FCS in Medium 199. The grafts and the cryoprotectant, were incubated at 37.degree. C. for two hours and were then frozen at -1.degree. C. per minute to -70.degree. C. and then stored in liquid nitrogen. After fast thawing of the grafts, their viability was determined by culturing for two weeks and by grafting to hairless mice. A final evaluation was made by grafting to three patients undergoing tattoo excision.
Johnstone et al. "Cryopreservation of Rabbit and Cat Corneas at -18.degree. to -24.degree. C.," Cornea, 11(3): 211-220 (1992), is directed to a simple procedure for cryopreservation of rabbit and cat corneas which utilizes a domestic freezer rather than liquid nitrogen or very low temperature freezers. Perfusion of cryopreservative is obtained by placing corneas in successive solutions of 50% fetal calf serum and McCarey-Kaufman medium with increasing glycerol and glucose content.
Using prior art methods, it is not possible to cryopreserve cultured tissue equivalents, in part because they are relatively thick and of heterogeneous cell layers. One of the functions of these tissues in vivo are to provide a permeability barrier. Tissue functions have to be considered in the development of a cryopreservation protocol. The present inventors have discovered a method for cryopreservation that is applicable to a number of cultured tissue equivalents and to mammalian skin, one that is a surprisingly effective and commercially practical method of cryopreservation.